Inserting a peptide tag into a protein facilitates the characterization of that protein when antibodies to the protein are not available. Recent techniques incorporate a peptide tag into a recombinant protein to aid protein purification or isolation of that protein. Antibodies recognizing the peptide tag facilitate purification and/or isolation (Brizzard, et al. BioTechniques 16:730-735, 1994; Fulton, et al. Eur. J. Immunol. 25:2069-2076, 1995; Olah, et al., Anal. Biochem. 221:94-102, 1994; Prickett, K. S., et al. BioTechniques 7:580-589, 1989). These methods usually depend upon the availability of unique restriction endonuclease recognition sites for the insertion of the antigenic peptide tag.
There are two basic strategies for incorporating an antigenic peptide inframe into a protein. In one method, the nucleic acid sequence encoding the peptide is added to the N-terminus equivalent of nucleic acid that encodes a protein. If the N-terminus contains a signal peptide that is cleaved when the protein enters the endoplasmic reticulum, then a tag inserted at this site will be cleaved during protein maturation. The peptide tag will not be available for mature protein isolation (Walter, P. et al. Annu. Rev. Cell Biol. 10:87-119, 1994).
C-terminal tagging techniques are also available (Olah Z., et al. Anal. Biochem 221:94-102, 1994 and Prickett, et al. BioTechniques 7:580-9, 1989). These techniques can be used when the addition of the tag to the C-terninus of a protein does not negatively affect the conformation of that protein. There are proteins where N-terminal and C-terminal tagging methods are not useful strategies for incorporating antigenic tags into a protein because, for example, the N-terminus is cleaved during protein maturation or additions to the C-terminus interfere with protein folding.
Reliance on restriction endonuclease recognition sites is not always practical and the addition of a peptide tag to the N-terninus or the C-terminus of a protein may not be useful. There is a need for a strategy to circumvent the limitations created by relying on restriction recognition sites and the limitations created when a tag is merely added to a terminal portion of a protein.
Varicella Zoster Virus (VZV) is a member of the Herpesvirus family. The VZV virion is formed as an icosahedral nucleocapsid surrounded by a lipid envelope containing a number of viral glycoproteins. The VZV envelope glycoproteins include gE, gB, gH, gI, gC and gL. gE is the most abundant protein in the virion envelope (Grose, et al. Infect. Immun. 40:381-388, 1983) and is encoded by VZV gene 68. The mature protein is about 98 kDa and is about 623 amino acid residues in length. Glycoprotein gE is the predominant immunogen of VZV and was formerly called gpI or gp98 (Grose, C. Annu. Rev. Microbiol. 44:59-80, 1990; Montalvo, E. A., et al. J. Virol. 53:761-770, 1985; and Yao, Z., et al. J. Virol. 67:305-314, 1993). gE induces neutralizing antibodies and the most antigenic fragment within gE is reported to be between residues 1-134. (Fowler, et al. Virology 214:531-540, 1995).